U.S. patent application number 09/841506 was filed with the patent office on 2002-12-05 for inhibition of cytokine generation.
Invention is credited to Kreutner, William, Schleimer, Robert P., Schroeder, John.
Application Number | 20020183344 09/841506 |
Document ID | / |
Family ID | 25285055 |
Filed Date | 2002-12-05 |
United States Patent
Application |
20020183344 |
Kind Code |
A1 |
Schleimer, Robert P. ; et
al. |
December 5, 2002 |
Inhibition of cytokine generation
Abstract
Methods of inhibiting the generation of pro-inflammatory
cytokines such as IL-4 and IL-13 in a human patient in need of such
inhibiting are disclosed.
Inventors: |
Schleimer, Robert P.;
(Baltimore, MD) ; Schroeder, John; (Baltimore,
MD) ; Kreutner, William; (West Paterson, NJ) |
Correspondence
Address: |
SCHERING-PLOUGH CORPORATION
PATENT DEPARTMENT (K-6-1, 1990)
2000 GALLOPING HILL ROAD
KENILWORTH
NJ
07033-0530
US
|
Family ID: |
25285055 |
Appl. No.: |
09/841506 |
Filed: |
April 24, 2001 |
Current U.S.
Class: |
514/290 |
Current CPC
Class: |
Y10S 514/853 20130101;
A61K 31/473 20130101; Y10S 514/887 20130101; Y10S 514/854
20130101 |
Class at
Publication: |
514/290 |
International
Class: |
A61K 031/473 |
Claims
We claim:
1) A method of blocking and/or inhibiting generation of
pro-inflammatory cytokines in a patient in need of such blocking
and/or inhibiting which comprises administering to such a patient
an effective amount of desloratadine.
2) The method of claims 1 wherein the pro-inflammatory cytokines
are IL-4 and IL-13.
3) A method of inhibiting generation of IL-4 and IL-13 from human
basophils in a patient exhibiting the symptoms of an allergic
and/or inflammatory condition which comprises administering to such
a patient an amount of desloratadine effective to inhibit the
generation of IL-4 and IL-13 and to concurrently treat the symptoms
of such an allergic and/or inflammatory condition.
4) A method of treating a disease state that has an allergic
component and an inflammatory component which comprises
administering to a patient in need of such treatment an amount of
desloratadine effective to produce an anti-inflammatory effect.
5) The method of claim 4 wherein the disease state is allergic
rhinitis, allergic asthma, atopic dermatitis, intestinal food
allergies, or urticaria.
6) A method of treating and/or preventing an allergic and/or
inflammatory condition and inhibiting secretion of IL-4 and IL-13
in a patient in need of such treating and/or preventing which
comprises administering to such a patient an effective amount of
desloratadine.
7) A method of treating IL-4 and IL-13 mediated disease states in a
patient in need of such treating which comprises administering to
such a patient an effective amount of desloratadine.
8) A method of claim 7 wherein effective amount of desloratadine is
sufficient to inhibit generation of IL-4 and IL-13 from human
basophils.
9) A method of inhibiting generation of IL-4 and IL-13 from human
basophils in a patient exhibiting the symptoms of an allergic
and/or inflammatory condition of the skin, eyes, intestinal tract,
and/or the upper and lower airway passages which comprises
administering to such a patient an amount of desloratadine
effective to inhibit the generation of IL-4 and IL-13 and to
concurrently treat the symptoms of such an allergic and/or
inflammatory condition.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to methods of using
desloratadine to inhibit generation of pro-inflammatory cytokines,
e.g., IL-4 and IL-13.
[0002] Mast cells and basophils play a role in allergic and
inflammatory diseases. These cells produce a wide variety of
mediators such as prostaglandins, e.g., prostaglandin D2,
leukotrienes, e.g., leukotriene C.sub.4 cytokines and histamine.
Cytokines are polypeptides secreted by cells that affect the
function of other cells. Cytokines, including interleukins, differ
widely in the types of cells affected and in biological activities
exhibited. Desloratadine, a non-sedating antihistamine, is
disclosed by Lippert, U., et al, Experimental Dermatology, 1995,
Vol. 4, 272-276 to be active in vitro in inhibiting the release of
the cytokines IL-6 by up to 40% and IL-8 by up to 50% from human
mast and basophilic cell lines. Human Fc.epsilon. RI.sup.+ cells
play a considerable pro-inflammatory role through the release of
histamine, tryptase and chymase. However, since human mast and
basophilic cell lines are defective in signaling through the high
affinity immunoglogulin-E(IgE) receptor, Fc.epsilon. RI, it is
difficult to predict what effect, if any, desloratadine has on the
IgE-mediated release of IL-6 and IL-8. . S. Molet, et al., Clinical
and Experimental Allergy, 1997, Vol. 27, pages 1167-1174 disclose
that desloratadine reduces histamine-induced release of IL-6 and
IL-8 in an in vitro study in endothelial cells. Kleine-Tebbe, J.,
et al, J. Allergy Clin. Immunol. 1994, Vol. 93, No. 2, pages
494-500 disclose that desloratadine inhibits IgE- and
non-IgE-mediated histamine release in an in vitro study in human
basophilic leukocyte cells.
[0003] Human basophils are a major source of the cytokines, IL-4
and IL-13 that are produced in vitro in mixed leukocyte cultures.
Production of IL-4 and IL-13 by basophils may play a role in
modulating a variety of activities that are involved in the
pathogenesis of allergic inflammatory conditions. For example, IL-4
and IL-13 each induce secretion of IgE- and IgG-4 by human B-cells.
(See Schroeder, J. T.,"The role of basophil-cytokine networks in
asthma." In Asthma and Allergic Diseases: Physiology,
Immunopharmacology, and Treatment, Editors: G. Marone, K. F.
Austen, S T Holgate, A. B. Kay, L. M. Lichtenstein, Academic Press,
San Diego, 75-84, 1998a). However, there is a no information on the
effect of desloratadine on the generation of cytokines such as IL-4
and IL-13 in basophils or any other cell types. One study by Gibbs,
et al. Naunyn Schmiedebegs Arch. Pharmacol ,1998, Vol. 357: 573-578
reports moderate (50%) inhibition of IL-4 and IL-13 secretion in
vitro in basophilic cells using relatively high concentrations of
terfenadine, a non-sedating antihistamine. This study did not test
whether the terfenadine concentrations used were toxic. The FDA has
withdrawn the two terfenadine NDAs because of the finding that
terfenadine was no longer safe for cardiac reasons for use in
treating seasonal allergic rhinitis.(Federal Register, Oct. 5,1998,
Vol. 63, page 53444).
[0004] There is a need for a safe, effective therapy to inhibit
secretion of pro-inflammatory cytokines such as IL-4 and IL-13 to
treat disease states, for example, allergic and/or inflammatory
conditions..
SUMMARY OF THE INVENTION
[0005] The present invention provides a method of inhibiting
generation of IL-4 and IL-13 in a human patient in need of such
inhibiting which comprises administering to such a patient an
effective amount of desloratadine.
[0006] Typically. IL-4 and IL-13 is generated from human basophils
as well as other cells, e.g., human B-cells.
[0007] The present invention also provides a method of inhibiting
generation of IL-4 and IL-13 from human basophils in a patient
exhibiting the symptoms of an allergic and/or inflammatory
condition which comprises administering to such a patient an amount
of desloratadine effective to inhibit the generation of IL-4 and
IL-13 and to concurrently treat the symptoms of such an allergic
and/or inflammatory conditions.
[0008] The present invention also provides a method of blocking
generation of pro-inflammatory cytokines in a patient in need of
such blocking which comprises administering to such a patient an
effective amount of desloratadine.
[0009] The present invention also provides a method of inhibiting
secretion of pro-inflammatory cytokines from human basophils in a
patient in need of such inhibiting which comprises administering to
such a patient an effective amount of desloratadine.
[0010] The preferred pro-inflammatory cytokines are IL-4 and
IL-13.
[0011] The patients in need of such inhibiting are those having
symptoms of allergic/inflammatory conditions of the airway
passages, skin, eyes and intestinal tract.
[0012] The present invention also provides a method of treating a
patient exhibiting the symptoms of allergiclinflammatory conditions
of the skin, eyes, intestinal tract,and/or upper and lower airway
passages which comprises administering to such a patient an
effective amount of desloratadine.
[0013] The patients in need of such inhibiting are those having
symptoms of an allergic and/or inflammatory condition of the skin,
eyes, intestinal tract, and/or the upper and lower airway
passages.
[0014] The present invention also provides a method of treating a
disease state that has an allergic component and an inflammatory
component which comprises administering to a patient in need of
such treatment an amount of desloratadine effective to produce an
anti-inflammatory effect.
[0015] The present invention also provides a method of treating
and/or preventing allergic asthma and inhibiting secretion of IL-4
and IL-13 in a patient in need of such treating which comprises
administering to such a patient an effective amount of
desloratadine.
[0016] The present invention also provides a method of treating
and/or preventing allergic rhinitis and inhibiting secretion of
IL-4 and IL-13 in a patient in need of such treating which
comprises administering to such a patient an effective amount of
desloratadine.
[0017] The present invention also provides a method of treating
and/or preventing atopic dermatitis and inhibiting secretion of
IL-4 and IL-13 in a patient in need of such treating which
comprises administering to such a patient an effective amount of
desloratadine.
[0018] The present invention also provides a method of treating
and/or preventing urticaria and inhibiting secretion of IL-4 and
IL-13 in a patient in need of such treating which comprises
administering to such a patient an effective amount of
desloratadine.
[0019] The present invention also provides a method of treating
IL-4 and IL-13 mediated disease states in a patient in need of such
treating which comprises administering to such patients an
effective amount of desloratadine to inhibit generation of IL-4 and
IL-13.
[0020] The effective amount of desloratadine is an amount
sufficient to inhibit and preferably block generation of IL-4 and
IL-13 from human cells.
[0021] The present invention also provides a method of inhibiting
generation of IL-4 and IL-13 from human basophils in a patient
exhibiting the symptoms of an allergic and/or inflammatory
condition of the skin, eyes, intestinal tract, and/or the upper and
lower airway passages which comprises administering to such a
patient an amount of desloratadine effective to inhibit the
generation of IL-4 and IL-13 and to concurrently treat the symptoms
of such an allergic and/or inflammatory condition.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIGS. 1a and 1b illustrate the effect of desloratadine on
histamine, leukotriene C.sub.4 ("LTC.sub.4") and IL-4 secretion by
human basophils.
[0023] FIG. 2 graphically illustrates the effect of desloratadine
on histamine release and IL-4 secretion in response to activation
by ionomycin.
[0024] FIG. 3 graphically illustrates the effect of desloratadine
on IL-13 secretion by human basophils activated by anti-IgE and
ionomycin.
[0025] FIG. 4 graphically illustrates that desloratadine inhibits
IL-13 secretion from basophils activated with IL-3 and PMA.
[0026] FIGS. 5a, 5b, 5c and 5d graphically illustrate the
inhibition of IL-4 mRNA expression in basophils pretreated with
desloratadine.
DETAILED DESCRIPTION OF THE INVENTION
[0027] In accordance with the methods of the present invention, we
demonstrated the ability of desloratadine to inhibit the generation
of IL-4 and IL-13 from human basophils while concurrently comparing
its efficacy in preventing mediator release from these cells using
a variety of stimuli. Desloratadine was found to be remarkably
(nearly 6-7) times more potent in reducing the secretion of IL-4
and IL-13 from human basophils induced by anti-IgE than it was at
inhibiting the mediators, histamine and LTC.sub.4, released in the
same culture supernatants. The cytokines, IL-4 and IL-13, were
equally inhibited by desloratadine following activation with
ionomycin despite the lack of an effect on the histamine release
induced with ionomycin. Desloratadine had a lesser effect at
inhibiting the IL-13 secreted in response to IL-3 and PMA,
suggesting that the antihistamine differentially targets individual
pathways of cytokine generation. Finally, there was no evidence
that desloratadine was cytotoxic, i.e., that it mediated its
inhibitory effects by causing decreased cell viability. In
accordance with the present invention, IL-4 mRNA accumulation was
also remarkably inhibited, by as much as 80%, following
pretreatment with desloratadine, suggesting that desloratadine
targets signals regulating cytokine gene transcription in addition
to those controlling mediator release.
[0028] The phrase "an allergic and/or inflammatory condition" means
those allergic and inflammatory conditions and symptoms found on
the skin,eyes, intestinal tract and/or in the upper and lower
airway passages from the nose to the lungs. Typical allergic and/or
inflammatory conditions of the skin or upper and lower airway
passages include seasonal and perennial allergic rhinitis,
non-allergic rhinitis, asthma including allergic and non-allergic
asthma, sinusitis, colds, dermatitis, especially allergic and
atopic dermatitis, and urticaria and symptomatic dermographism.
Typical allergic and/or inflammatory conditions of the eyes
include, but are not limited to, allergic conjunctivitis. Typical
allergic and/or inflammatory conditions of the intestinal tract,
but are not limited to, food allergies.
[0029] The term "human" as used herein includes a male or female
pediatric subject of less than 12 years of age to less than 18
years of age, a male or female pediatric subject of greater than 12
years of age to less than 18 years, and male and female adults of
18 years of age and older.
[0030] The term "pro-inflammatory cytokines" as used herein means
those cytokines associated with allergic and inflammatory reactions
of the skin, eyes, intestinal tract, and airway passages of humans
exposed to allergens. Typically suitable pro-inflammatory cytokines
include IL-3, IL-4, IL-5, IL-6, IL-8, IL-9 and IL-13.
[0031] The amount of desloratadine effective for treating or
preventing allergic and inflammatory conditions of the skin or
airway passages will vary with the age, sex, body weight and
severity of the allergic and inflammatory condition of the patient.
Typically, the amount of desloratadine effective for treating or
preventing such allergic and inflammatory conditions in an adult
human of age older than 12 is in the range of about 2.5 mg/day to
about 45 mg/day, preferably about 2.5 mg/day to about 20 mg/day, or
about 5.0 mg/day to about 15 mg/day, or about 5.0 mg/day to about
10 mg/day, more preferably about 5.0 mg/day to about 7.5 mg/day,
and most preferably about 5.0 mg/day in single or divided doses,
e.g., 2.times.2.5 mg/day, or a single dose of 5.0 mg/day.
[0032] Desloratadine is a non-sedating long acting histamine
antagonist with potent and selective peripheral H1-receptor
antagonist activity. In vitro and in vivo animal pharmacology
studies have been conducted to assess various pharmacodynamic
effects of desloratadine and loratadine. In assessing central
nervous system ("CNS") activity in mice, desloratadine was
relatively free of producing alterations in behavior, neurologic or
autonomic function. The potential for desloratadine to occupy brain
H1-receptors was assessed in guinea pigs following IP
administration and results suggest poor access to central histamine
receptors for desloratadine.
[0033] The clinical efficacy and safety of desloratadine has been
documented in over 3,200 seasonal allergic rhinitis patients in 4
double-blinded, randomized clinical trials. The results of these
clinical studies demonstrated the efficacy of desloratadine in the
treatment of adult and adolescent patients with seasonal
rhinitis.
[0034] Efficacy endpoints in all the studies were Total Symptom
Score, Total Nasal Symptom Score, Total Non-nasal Symptom Score,
and Health Quality of Life (HQOL) analysis in efficacy trials.
Desloratadine (5 mg once daily) significantly reduced the total
symptom scores (the sum of individual scores for rhinorrhea,
sneezing, congestion/stuffiness, nasal itching, itchy/burning eyes,
tearing, ocular redness, and itchy ears/palate). Desloratadine (5
mg) was significantly (p<0.01) more effective than placebo in
reducing nasal symptoms. An important efficacy endpoint analyzed in
the desloratadine studies is the AM NOW total symptom score. This
parameter measures the total symptom relief by the patient after 24
hours before taking the next day dose. Statistically significant
(p<0.05) reductions were maintained for the full 24 hour dosing
interval over the entire 5 mg to 20 mg dosage range
[0035] There were no significant differences in the effectiveness
of desloratadine (over the entire 5 mg to 20 mg dosage range)
across subgroups of patients defined by gender, age, or race.
Desloratadine is particularly useful for the treatment and
prevention of the nasal (stuffiness/congestion, rhinorrhea, nasal
itching, sneezing) and non-nasal (itchy/burning eyes,
tearing/watery eyes, redness of the eyes, itching of the
ears/palate) symptoms of seasonal allergic rhinitis, including
nasal congestion, in patients in need of such treating and/ or
preventing.
[0036] U.S. Pat. No. 4,659,716 discloses methods of making
desloratadine, pharmaceutical compositions containing it and
methods of using desloratadine and pharmaceutical compositions
containing it to treat allergic reaction in mammals.
[0037] U.S. Pat. No. 5,595,997 discloses pharmaceutical
compositions containing desloratadine and methods of using
desloratadine for treating and preventing various disease states,
e.g., allergic rhinitis.
[0038] U.S. Pat. No. 6,100,274 discloses stable pharmaceutical
compositions containing desloratadine suitable for oral
administration to treat allergic reactions, e.g., allergic
rhinitis.
[0039] The pharmaceutical compositions of desloratadine can be
adapted for any mode of administration e.g., for oral, parenteral,
e.g., subcutaneous ("SC"), intramuscular ("IM"), and
intraperitoneal ("IP"), topical or vaginal administration or by
inhalation (orally or intranasally). Preferably desloratadine is
administered orally.
[0040] Such pharmaceutical compositions may be formulated by
combining desloratadine or an equivalent amount of a
pharmaceutically acceptable salt thereof with a suitable, inert,
pharmaceutically acceptable carrier or diluent that may be either
solid or liquid. Desloratadine may be converted into the
pharmaceutically acceptable acid addition salts by admixing it with
an equivalent amount of a pharmaceutically acceptable acid.
Typically suitable pharmaceutically acceptable acids include the
mineral acids, e.g., HNO.sub.3, H.sub.2SO.sub.4, H.sub.3PO.sub.4,
HCl, HBr, organic acids, including, but not limited to, acetic,
trifluoroacetic, propionic, lactic, maleic, succinic, tartaric,
glucuronic and citric acids as well as alkyl or arylsulfonic acids,
such as p-toluenesulfonic acid, 2-naphthalenesulfonic acid, or
methanesulfonic acid. The preferred pharmaceutically acceptable
salts are trifluoroacetate, tosylate, mesylate, and chloride.
Desloratadine is more stable as the free base than as an acid
addition salt and the use of the desloratadine free base in
pharmaceutical compositions of the present invention is more
preferred. See U.S. Pat. No. 6,100,274.
[0041] Solid form preparations include powders, tablets,
dispersible granules, capsules, cachets and suppositories. The
powders and tablets may be comprised of from about 2.5 to about 95
percent active ingredient preferably from about 2.5 to about 20
percent, more preferably from about 2.5 to about 10 percent, or
from about 2.5 to about 5 percent and most preferably 5 percent of
the active ingredient. Suitable solid carriers are known in the
art, e.g. magnesium carbonate, magnesium stearate, talc, sugar or
lactose. Tablets, powders, cachets and capsules can be used as
solid dosage forms suitable for oral administration. Examples of
pharmaceutically acceptable carriers and methods of manufacture for
various compositions may be found in A. Gennaro (ed.), Remington's
Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co.,
Easton, Pa.
[0042] Liquid form preparations include solutions, suspensions and
emulsions. As an example may be mentioned water or water-propylene
glycol solutions for parenteral injection. Solid form preparations
may be converted into liquid preparations shortly before use for
either oral or administration. Parenteral forms to be injected
intravenously, intramuscularly or subcutaneously are usually in the
form of sterile solutions and may contain tonicity agents (salts or
glucose), and buffers. Opacifiers may be included in oral
solutions, suspensions and emulsions. Liquid form preparations may
also include solutions for intranasal administration. The liquid
form preparations may comprise the same ranges of active ingredient
is as used in solid form preparations.
[0043] Aerosol preparations suitable for inhalation may include
solutions and solids in powder form, which may be in combination
with a pharmaceutically acceptable carrier, such as an inert
compressed gas, e.g., nitrogen.
[0044] Also included are solid form preparations which are intended
to be converted, shortly before use, to liquid form preparations
for either oral or parenteral administration. Such liquid forms
include solutions, suspensions and emulsions.
[0045] The compounds of the invention may also be deliverable
transdermally. The transdermal compositions can take the form of
creams, lotions, aerosols and/or emulsions and can be included in a
transdermal patch of the matrix or reservoir type as are
conventional in the art for this purpose.
[0046] Preferably, the pharmaceutical preparation is in a unit
dosage form. In such form, the preparation is subdivided into
suitably sized unit doses containing appropriate quantities of the
active component, e.g., an effective amount to achieve the desired
purpose.
[0047] Further, desloratadine may be administered in association
with therapeutically effective amounts of steroids, e.g. mometasone
furoate, beclomethasone dipropinate or fluticasone propinate,
leukotriene inhibitors, e.g., montelukast sodium or zafirlukast,
and/or an upper airway passage decongestant including, but not
limited to phenylephedrine, pseudoephedrine and phenylpropanolamine
or pharmaceutically acceptable salts thereof, in accordance with
the dosing levels known to those skilled in the art and as
described in the Physicians' Desk Reference. The use of the upper
airway passage decongestant, pseudoephedrine HCl, is preferred.
EXAMPLES
MATERIALS AND METHODS
[0048] Special Reagents
[0049] All reagents were purchased unless otherwise noted.
Piperazine-N,N'-bis-2-ethanesulfonic acid (PIPES), ionomycin, FMLP,
PMA, and fetal bovine serum (FBS) from Sigma Chemical Co. (St.
Louis, Mo.); RPMI-1640 and Iscove's modified Dulbecco's medium
(IMDM) both with L-glutamine and containing 25 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesu- lfonic acid (HEPES),
gentamicin, nonessential amino acids (100.times.) from Life
Technologies, Inc., Grand Island, N.Y.); and Percoll from Pharmacia
(Piscataway, N.J.). The desloratadine used in these experiments was
supplied by The Schering-Plough Research Institute. A 0.1 M stock
solution was made in DMSO, aliquoted, and frozen at -20.degree. C.
All pipes-containing buffers were made from stock 10X PIPES (250 mM
PIPES, 1.20 M NaCl, and 50 mM KCL, pH 7.3 and stored at 4.degree.
C.). Isotonic Percoll, (referred to as 100% Percoll) was made by
mixing 9 parts Percoll with 1 part 10X PIPES. PAG contained
one-tenth 10X PIPES. 0.003% HAS, and 0.1% D-glucose. PAG-EDTA
additionally contained 4 mM EDTA. Percoll solutions used for cell
isolation were all made by mixing the appropriate amounts of 100%
Percoll with lX PIPES.
[0050] Cell Preparation and Culture
[0051] Mixed leukocyte suspensions containing basophils were
prepared either using double-Percoll density centrifugation as
described (Schroeder, et al., J. Immunol.,1994, Vol. 153: 1808, or
by a combination of countercurrent elutriation and Percoll density
centrifugation protocols (MacGlashan, et al., J. Immunol.,1994,
Vol. 153:3006-3016). The percentages of basophils obtained using
these protocols typically ranged between 5-50% and 10-30%,
respectively, and were determined by counting Alcian blue positive
and negative stained cells on a Spiers-Levy chamber (Gilbert and
Ornstein, 1975). For some experiments, the basophils were
additionally purified to >99.9% using a negative-selection
protocol (Miltenyi Corp., Auburn, Calif.). For all experiments
other than those assessing IL-4 mRNA expression, the cells were
cultured in 96-well flat-bottom microtiter plates (in duplicate)
using IMDM supplemented with 5% heat-inactivated (56.degree. C. for
30 min.) FBS, 1.times.non-essential amino acids, and 5 .mu.g/ml
gentamicin (C-IMDM). For the analysis of IL-4 mRNA expression,
cells in C-IMDM were cultured in autoclaved (RNase-free) 1.5 ml
microcentrifuge tubes (see below), since this allowed for a more
precise way to quantitatively isolate mRNA without the need for
transferring cells from culture wells before extraction. For each
condition, leukocyte suspensions containing approximately
100,000-500,000 basophils in 100 .mu.l of C-IMDM were prewarmed to
37.degree. C. before adding 100 .mu.l of desloratadine
concentrations in C-IMDM also prewarmed to 37.degree. C. After 15
minutes preincubation, the cells were then activated by adding 50
.mu.l of 5.times.(5 times the final concentration) of stimulus. It
is important to note here that the concentrations of stimuli used
were optimal for IL-4 generation rather than mediator release. This
is particularly true for anti-IgE antibody, which has been shown to
induce IL-4 at concentrations 10-fold less than those causing
optimal histamine release (Schroeder, et al., ibid). For
harvesting, cultures were centrifuged and cell-free supernatants
collected for mediator release and cytokine analysis, as described
(Schroeder, et al., ibid). Histamine, LTC.sub.4, and IL-4 were all
measured in aliquots of culture supernatant taken at 4 hours. For
histamine, this meant taking 20-50 .mu.l of supernatant and
diluting it in 1 ml of PAG buffer containing 1.6% HClO.sub.4. After
an overnight precipitation at 4.degree. C., the samples were
assayed by automated fluorimetry (Siraganian,R. P., Anal. Biochem..
1974, Vol.57:383-394). LTC.sub.4 was measured by an in-house RIA
(MacGlashan, et al., J. Immunol. 1986, Vol.136:2231-2239). IL-4
protein was measured by a commercial ELISA (Biosource
International). Culture supernatants were harvested after 20 hours
incubation for all experiments investigating the effects of
desloratadine on IL-13 secretion. In some instances, IL-4 was also
measured at this time point, particularly when ionomycin was used,
since the kinetics for the secretion of this cytokine extend beyond
4 hours with this stimulus (Schroeder, et al., J. Leuk. Biol.,
1998, Vol. 63:692-698). IL-13 protein measurements were also made
using a commercial ELISA (Immunotech).
[0052] RNA Isolation and Semi-Quantitative Analysis of IL-4 mRNA
Expression
[0053] Cultures for the analysis of IL-4 mRNA were performed in 1.5
ml polypropylene microcentrifuge tubes, as described above. Cells
were pretreated with desloratadine (10 .mu.M) for 15 minutes prior
to activating with anti-IgE antibody (10-20 ng/ml). Total RNA was
isolated using the RNAzol protocol (Tel-test Inc., Friendswood,
Tex.) after 2 hours incubation, which is the time that IL-4 message
expression peaks using IgE-dependent activation (Schroeder, et al.,
J. Immunol..1997, Vol.158:5448-5454). Following isopropanol
precipitation, the RNA was washed with 70% ethanol and dried under
vacuum. Subsequently, the RNA was resuspended in 25 .mu.l of
diethipyrocarbonate (DEPC)-treated water and stored at -80.degree.
C. Reverse Transcription (RT) and polymerase chain reaction (PCR)
were performed with serial dilutions of RNA as previously detailed
(MacGlashan, et al., J. Immunol, 1994; Vol. 152:3006-3016;
Schroeder, at al., J. Immunol., 1997, Vol. 158: 5448-5454) using
the GeneAmp RNA PCR kit (Perkin-Elmer Cetus, Norwalk, Conn.). PCR
products were visualized in 3% agrose gels using ethidium bromide
staining. As noted elsewhere, two distinct bands for IL-4 were
observed. A dominant band was seen with a size of approximately 460
bp. The source of the smaller, fainter band is uncertain, but is
thought to be an alternatively spliced form of IL-4 (Atamas, et
al., J. Immunol, 1996, Vol.156:435-441). The two bands are
routinely observed using either pure or enriched suspensions of
basophils.
[0054] FIGS. 1a and 1b graphically illustrate the effect of
desloratadine on histamine, LTC.sub.4, and IL-4 secretion by human
basophils. For FIG. 1a, mixed leukocyte suspensions containing
3-55% basophils were prepared from whole blood using Percoll
density centrifugation. Cells were pretreated 15 min. with the
indicated concentrations of desloratadine before activating with
anti-IgE antibody (10-20 ng/ml). All three products were measured
using the same culture supernatants harvested after 4 hours
incubation. Values are the mean.+-.SEM (n=5). Control release for
each product: Histamine: 40.+-.6% of total, IL-4: 403.+-.207
pg/10.sup.6 basophils, and LTC.sub.4: 860.+-.127 pg/10.sup.6
basophils. FIG. 1b shows the effect of desloratadine on histamine,
LTC.sub.4, and IL-4 secretion from human basophils, 99% purity.
Control release for histamine, LTC.sub.4, and IL-4 were 60% of
total, 903 pg/10.sup.6 basophils, and 854 pg/10.sup.6 basophils,
respectively.
[0055] FIG. 2 illustrates the effect of desloratadine on histamine
release (HR) and IL-4 secretion in response to ionomycin. Basophil
suspensions of 42, 99, and 98% purity were pretreated 15 min. with
0.1, 1.0, and 10 .mu.M desloratadine. Cells were then activated
with ionomycin (500 ng/ml) for 4 hours. Culture supernatants were
harvested and assayed for histamine and IL-4 protein. Values
represent the mean.+-.SEM, n=3. Control levels of IL-4 were 3398,
253, and 348 pg/10.sup.6 basophils. Percent histamine release in
the same cultures was 91, 25, and 10%, respectively.
[0056] FIG. 3 illustrates the effect of desloratadine on IL-13
secretion by human basophils activated by anti-IgE or ionomycin.
Basophil suspensions ranging in purity between 6-99% were
pretreated 15 min. with the indicated concentrations of
desloratadine. Cells were activated with either anti-IgE (10-20
ng/ml) or ionomycin (500 ng/ml). Culture supernatants were
harvested after 18 hours incubation and assayed for IL-13 protein
by ELISA. Values with error bars represent the mean.+-.SEM, n=3.
IL-13 protein in control cultures averaged 169.+-.35 and 334.+-.144
pg/10.sup.6 basophils for anti-IgE and ionomycin, respectively.
Values indicated by the open circles are the mean for two
experiments (control levels of IL-13 were 260 and 230 pg/10.sup.6
basophils).
[0057] FIG. 4 graphically illustrates that desloratadine inhibits
IL-13 secretion from basophils activated with IL-3 and PMA.
Basophils (up to 94% purity) were prepared from whole blood using
double Percoll density centrifugation and negative selection
protocols. Cells were pretreated with the indicated concentrations
of desloratadine before adding IL-3 (100 ng/ml) or PMA (2 ng/ml).
Culture supernatants were harvested after 18 hours incubation and
assayed for IL-13 protein by ELISA. Values represent the
mean.+-.SEM, n=3-4. The amount of IL-13 secretion in the absence of
desloratadine averaged 570.+-.142 pg/10.sup.6 basophils for IL-3
activation and 216.+-.120 pg/10.sup.6 basophils for PMA
induction.
[0058] FIGS. 5a-d graphically illustrate the inhibition of IL-4
mRNA expression in basophils pretreated with desloratadine. Cells
were incubated 15 min. with desloratadine (10 .mu.M) or DMSO
(1:10,000) before receiving anti-IgE (10 ng/ml) or medium alone.
Total RNA was isolated after 2 h and used for dilutional RT-PCR
analysis to compare message expression for IL-4 and the
housekeeping gene, hypoxanthine phosphoribosyl transferase (HPRT)
(panel a). Densitometric analysis is shown for undiluted RNA
expression (panel b). The histamine and IL-4 secretion in the
supernatants after 2b are shown for comparison (panels c and d,
respectively). Values represent the mean.+-.SEM, n=3.
DISCUSSION
[0059] In the first series of experiments (See FIGS. 1a& 1b),
we demonstrated the ability of desloratadine to block IL-4
secretion from basophils triggered by anti-IgE, and compared this
inhibition to that seen for histamine and LTC.sub.4 release in the
same 4 h culture supernatants. As shown in FIG. 1a, desloratadine
dose-dependently inhibited the release of histamine and LTC.sub.4
at concentrations between 1 .mu.M (10.sup.-6 M) and 10 .mu.M
(10.sup.-5 M). For comparison, desloratadine was strikingly more
potent against IL-4 secretion than for preformed mediator release.
Cells pretreated with 1 .mu.M to 10 .mu.M desloratadine secreted
40% to 80% less IL-4 than cells not exposed to drug. Nearly a
seven-fold greater concentration of desloratadine was necessary to
cause a comparable inhibition in the release of histamine and
LTC.sub.4 by these cells. The inhibition of IL-4, unlike that seen
for histamine, appeared to plateau in the micromolar range of
desloratadine and then reached progressively higher values at
concentrations between 1 .mu.M and 10 .mu.M desloratadine. As shown
in FIG. 1b, this effect was also evident using basophils purified
to greater than 99%, indicating that desloratadine was directly
affecting basophils for the inhibition of IL-4. Desloratadine also
inhibited histamine from pure basophils much as it did for impure
suspensions.
[0060] We tested whether desloratadine affected IL-4, histamine,
and LTC.sub.4 release from basophils activated by other stimuli.
FIG. 2 shows that the secretion of histamine induced by the calcium
ionophore, ionomycin, was unaffected by desloratadine at
concentrations of 0.1, 1.0, and 10 .mu.M. However, the high levels
of IL-4 that were secreted in response to ionomycin stimulation
were remarkably inhibited by the same concentrations of
desloratadine (i.e. 0.1, 1.0 and 10 .mu.M). The histamine and
LTC.sub.4 made in response to FMLP stimulation was also not
inhibited by desloratadine (data not shown).
[0061] Redrup, et al., J.Immunol., 1998, Vol.160:1957-1964 reported
that (1) multiple stimuli induce pharmacologically distinct
pathways for the generation of IL-13 in human basophils and that
(2) both anti-IgE and ionomycin induced IL-13 secretion that was
sensitive to the immunosuppressive drug, FK-506, suggesting that
these stimuli utilize a calcineurin-dependent pathway to generate
this cytokine. In the Redrup, et al study, the IL-13 made in
response to IL-3 or PMA activation was unaffected by FK-506, and
only protein generated in response to the phorbol ester was
inhibited by PKC inhibitors. Therefore, we addressed whether the
IL-13 induced by these stimuli is also differentially affected with
desloratadine pretreatment. FIG. 3 shows that IL-13 generated in
response to either anti-IgE or ionomycin. In fact, there was
remarkable similarity in the shape of the concentration-response
curve of desloratadine for inhibition of IL-13 induced by either
stimulus. These curves in FIG. 3 were nearly identical to that seen
for the inhibition of IL-4 by desloratadine as shown in FIGS. 1 and
2. In particular, an initial plateau was seen at 1 .mu.M at the
level of 50-70% inhibition. Dose-dependent inhibition of IL-13
continued as desloratadine concentrations were increased up to 10
.mu.M. Interestingly, the response to desloratadine pretreatment
was considerably different in two experiments using impure (6-11%)
basophil suspensions. With these cell preparations, desloratadine
was considerably less effective in preventing the IL-13 secreted in
response to anti-IgE, requiring nearly 10-fold greater
concentrations to produce the same IC.sub.50 seen using more pure
basophil suspensions. FIG. 4 shows that desloratadine also
inhibited the IL-13 made in response to IL-3 or PMA, albeit to a
lesser extent and with inhibition curves that appeared more
log-linear. In fact, the drug inhibited IL-3-mediated IL-13
production with an IC.sub.50 of approximately 2 .mu.M, which was
nearly 5-fold that necessary to inhibit the IL-13 secreted with
anti-IgE or ionomycin activation. Desloratadine inhibited the IL-13
induced by PMA in similar fashion, with inhibition seen between 1
.mu.M and 10 .mu.M concentrations of drug.
[0062] Unlike histamine and LTC.sub.4, both of which are released
from basophils within minutes after activation, IL-4 protein is
first detected after 1-2 hours and IL-13 some 48 hours after
activation. Therefore, it seemed possible that the kinetics of
mediator release vs. cytokine production might account for the
differences in their sensitivity to desloratadine, particularly if
cell lysis was a factor. However, we saw no evidence that 10 .mu.M
desloratadine, even when combined with stimulus for 18 hours, was
cytotoxic, i.e., caused cell death as assessed by trypan blue
exclusion in pure basophil cultures (data not shown). Cells did
stain with trypan blue when cultured with 100 .mu.M desloratadine
alone. Furthermore, histamine was detected in these cultures,
indicating that this concentration of desloratadine caused cell
lysis.
[0063] Finally, to investigate the possibility that desloratadine
inhibits cytokine secretion from basophils by affecting the
accumulation of mRNA, we examined the effect of desloratadine (10
.mu.M) pretreatment on the expression of IL-4 message. For these
experiments, we used a dilutional RT-PCR protocol to provide a
semi-quantitative analysis. Panel a of FIG. 5 clearly shows that
IL-4 mRNA expression was induced in cells activated with anti-IgE
(lanes 8-10) relative to cells not receiving stimulus (lanes 2-4).
Pretreatment with desloratadine resulted in a substantial decrease
of the induced cytokine message (lanes 11-13), while having no
effect on the expression of the housekeeping gene, HPRT. Panel b of
FIG. 5 shows the average densitometric analysis of three
experiments, indicating that desloratadine pretreatment caused
approximately an 80% reduction in the IL-4 message accumulated with
anti-IgE activation. For comparison, desloratadine (10 .mu.M) also
inhibited the histamine and IL-4 protein secreted into the
supernatants of these cultures, as shown in panels c and d,
respectively, of FIG. 5.
[0064] Our findings support the concept that the mechanisms
underlying the efficacy of H1-antagonists in the treatment of
allergic rhinitis extend beyond their ability to prevent histamine
from binding to its H1 receptor by showing that desloratadine
possesses inhibitory activity against the generation of IL-4 and
IL-13 from human basophils. Others have shown that desloratadine
prevents IgE-mediated histamine release and LTC.sub.4 generation by
human basophils and our results confirm these findings ( See
Kleine-Tebbe, et al., 1994; and Genovese, et al., Clin. Exp.
Allergy, 1996, Vol. 27:559-567). However, in accordance with this
invention, we have shown that desloratadine is remarkably (6 to
7-fold) more effective at inhibiting IL-4 and IL-13 induced by
anti-IgE than it is at blocking histamine and LTC.sub.4 release in
these cultures. Interestingly, in accordance with this invention,
desloratadine inhibited IL-4 and IL-13 with a dose response curve
that plateaued at 1 and 10 .mu.M concentrations. (See FIG 1b.) This
effect was not seen for mediator release and occurred only for
cytokine (IL-4 and IL-13) produced in response to anti-IgE or
ionomycin. While the nature of this inhibition is presently
unknown, we predict that desloratadine targets at least two signals
along with common pathway triggered by both stimuli. Thus, at
concentrations below 1 .mu.M desloratadine, the inhibition may
result from disruption of a signal involved only in cytokine
generation, while at concentrations above 1 .mu.M, a second signal
may also be affected, resulting in inhibition of preformed mediator
release for most stimuli.
[0065] We found desloratadine to be less effective at preventing
mediator release induced by IgE-independent stimuli, such as
ionomycin, fmip, and phorbol ester. In fact, we found no evidence
that the histamine and/or LTC.sub.4 released with these stimuli
were inhibited by any of the desloratadine concentrations tested
(data not shown). In contrast, desloratadine was quite effective in
blocking the production of IL-4 induced by ionomycin and the IL-13
generated in response to either ionomycin, PMA, or IL-3. As noted
above, desloratadine equally inhibited the IL-4 and IL-13 induced
by ionomycin or anti-IgE with inhibition curves that plateaued at 1
and 10 .mu.M concentrations of drug. However, the same pattern and
potency of inhibition was not observed for the inhibition of IL-13
induced by IL-3 or PMA. With regard to cytokine production, both
anti-IgE and ionophore induced IL-4 and IL-13 that are inhibited by
the immunosuppressive drug, FK506, suggesting that these stimuli
utilize a calcium-dependent calcineurin pathway for the generation
of these cytokines. However, desloratadine does not block the IL-13
made in response to IL-3 or PMA, both of which are thought to
activate separate pathways. Thus, desloratadine may inhibit
IgE-dependent cytokine secretion and mediator release by simply
preventing changes in cytosolic calcium. This effect, however, does
not fully explain why desloratadine inhibited cytokine induced by
ionomycin while having no effect on mediator release. Since
IgE-crosslinking and Ca.sup.2+ ionophores appear to activate
calcineurin in basophils, it is possible that desloratadine targets
a factor within pathway initiated by this phosphatase.
[0066] By using pure basophil suspensions we were able to conclude
for the first time whether desloratadine mediates its inhibitory
effect on basophil mediator release and cytokine production
directly. We could detect no evidence, as assessed by trypan blue
exclusion, that the decreased IL-4 and IL-13 secreted was
accompanied by increased cell death during the 4-20 hour
incubations. This was true even with 10 .mu.M desloratadine which
caused nearly complete(>95%) inhibition of cytokine secretion.
Cell lysis was observed at a concentration of 100 .mu.M.
[0067] In accordance with the methods of the present invention, we
have demonstrated that desloratadine inhibits the accumulation of
IL-4 mRNA, supporting the concept that desloratadine can negatively
regulate factors important for cytokine gene transcription.
Interestingly, desloratadine pretreatment resulted in nearly 80%
inhibition of the IgE-mediated increase of IL-4 mRNA. However, the
secretion of protein for this cytokine (IL-4) in the same cultures
was below detection, and was thus inhibited >95%. Once again
this may imply that desloratadine targets multiple pathways in the
generation of cytokines, affecting more than just signals important
for gene transcription. In fact, it remains possible that
transcription is not at all affected but that IL-4 and IL-13 mRNA
is made unstable in some way and that this accounts for the
inhibitory action of desloratadine. If so, the instability must be
specific for cytokine mRNA, since our results demonstrated that
housekeeping gene (HPRT) expression is unaffected by
desloratadine.
[0068] We expect that inhibitory effects of desloratadine on
cytokine generation and mediator release seen in these in vitro
studies should translate into the clinical efficacy of
desloratadine against allergic and inflammatory conditions of the
skin, e.g., atopic dermatitis and urticaria, and in the eyes, e.g.,
allergic conjunctivitis, and in the intestinal tract, e.g., food
allergies, and in the upper and lower airway passage, e.g.,
allergic rhinitis, especially seasonal allergic rhinitis. Given the
fact IL-4 and IL-13 help regulate the synthesis of IgE, activate
endothelium for VCAM-1 -mediated eosinophil transmigration, and
that IL-4 promotes the development of the T-helper 2 phenotype, we
expect that desloratadine will mediate anti-allergic activity, not
only by inhibiting mediator release, but also by blocking the
generation of pro-inflammatory cytokines IL-3, IL-4, IL-5, IL-6,
IL-8, IL-9 and IL-13.
CLINICAL STUDIES
[0069] The following clinical studies are designed to show that
desloratadine provides combined antihistaminic and anti-allergic
inflammatory effects to control both early and late phase allergic
reactions and clinical symptoms on human subjects exposed to
allergens or exhibiting the signs and symptoms of allergic and/or
inflammatory diseases of the skin, eyes intestinal tract, and
airway passages.
STUDY NO. 1
[0070] Objective
[0071] To demonstrate that nasal allergen provocation of atopic
human subjects induces systemic allergic/immunologic effects, such
as, increased cytokine production by basophils and lymphocytes, and
that this will be manifested in vitro (increased cytokine release)
and in vivo (increase in late-phase reaction in allergen-induced
skin reaction).
[0072] Study Design
[0073] This will be an open-label, non-therapeutic, pilot study,
involving 6 to 10 asymptomatic atopic subjects with documented
histories of seasonal allergic rhinitis (and possibly allergic
asthma) who will be challenged nasally with allergen on 3
consecutive days.
[0074] Blood specimens will be collected 3 days before beginning
the nasal challenges, daily prior to each challenge, and one week
after the first challenge. Basophils and lymphocytes will be
isolated using standard Percoll gradient techniques and their RNA
will be extracted and tested using a real time PCR assay with
primers for IL-3, IL-4, IL-5, IL-6, IL-9 IL-13, IL-5 and
RANTES.
[0075] The mRNA for these cellular products will be quantitated by
comparison to the PCR product of a housekeeping gene (GAPDH).
Leukocyte differential, and total eosinophil and basophil counts
will be used to assess the potential effect of changes in cell
numbers on allergen-induced changes in cytokine/chemokine mRNA.
[0076] Dose-titrated allergen-induced skin wheal reaction testing
will be done at the time of initial blood drawing above (i.e., 3
days prior to the first nasal allergen challenge) and one week
after the last nasal allergen challenge.
[0077] Nasal symptom severity scores will be monitored during each
nasal allergen provocation day. It is expected that induction of
cytokine transcription will correlate to worsening nasal
symptomatology.
[0078] Primary Endpoint:
[0079] Based on in vitro data, the expected primary outcome should
be allergen-induced increases in inflammatory cells and
up-regulation of IL-4 and IL-13 transcription in the skin biopsy
tissue.
[0080] Secondary Endpoints:
[0081] Changes from baseline in peripheral leukocytes, including
eosinophils, basophils and lymphocytes.
[0082] Changes from baseline in cytokine production by
leukocytes.
[0083] Change from baseline in the size of the allergen-induced
skin wheal late phase reaction.
[0084] Correlation of the above changes with clinical
symptomatology.
STUDY NO. 2
[0085] Objective
[0086] Identification of the biochemical and clinical effects of
the anti-allergic and anti-inflammatory properties of
desloratadine.
[0087] A study of the effects of desloratadine on the induction of
peripheral blood leukocyte cytokines by nasal allergen provocation
in atopic human subjects: a preliminary clinical study to
demonstrate that desloratadine provides combined antihistaminic and
anti-allergic effects to control early and late phase allergic
reaction and clinical symptoms in human subjects exposed to nasal
allergen provocation. The number of subjects for Study No. 2 will
be determined based on the findings in the pilot in vitro and
clinical study..
[0088] Hypotheses
[0089] There is evidence that nasal allergen provocation of atopic
individuals induces systemic effects, e.g., peripheral blood
eosinophil counts are increased between 6 to 24 hours following
this procedure and consistent increases in lower airway
responsiveness, in patients with allergic rhinitis and asthma, have
followed nasal allergen provocation. It is possible that the
systemic effects of allergic rhinitis are mediated through
increased proliferation of peripheral leukocytes and increased
cytokine production by these cells. The underlying rationale for
these hypotheses is that these phenomena are dependent on the state
of activation in immune cells, including basophils and
lymphocytes.
[0090] Study Design
[0091] The clinical efficacy will involve the same essential nasal
allergen challenge design and blood specimen collections and
allergen-induced skin wheal testing as in Study No. (See below.)
Study No. 2, however, will be a double-blind, placebo-controlled,
cross-over design during which desloratadine (5 mg once daily) and
placebo will be taken daily for 5-7 days prior to the first blood
drawing and throughout the remainder of the testing period: the
repeat allergen-induced skin wheal test will occur one week after
the last nasal allergen challenge. The number of subjects for this
study will be determined by power analysis dependent on the results
of the preliminary study. A two-week washout period will intervene
between the two treatment periods.
1 Flow Chart (Study Days) -3 (baseline) 0 1 2 9 blood drawn nasal
nasal nasal skin wheal allergen allergen allergen test challenge**
challenge** challenge** skin wheal blood draw* blood draw* blood
draw* .fwdarw. dosing test dosing dosing dosing *for leukocyte
differential, total eosinophil and basophil counts for isolation of
basophils and lymphocytes and extraction of their RNA for PCR assay
(IL-4, IL-3, IL-13, IL-5, IL-6 and RANTES) **clinical symptoms and
severity scoring
[0092] Clinical Efficacy Endpoints
[0093] Comparisons of results will be made between treatment groups
and within treatment groups for:
[0094] Changes from baseline in peripheral leukocytes, including
eosinophils, basophils and lymphocytes.
[0095] Changes from baseline in cytokine production.
[0096] Changes from baseline in the size of the allergen-induced
skin wheal late phase reaction.
[0097] Correlation of the above changes with clinical
symptomatology.
[0098] Desloratadine is expected to provide combined anti-histamine
and anti-allergic inflammatory effects to control both early and
late phase allergic reactions and clinical symptoms in humans.
* * * * *